| Parkinson's disease(PD)is characterized by the progressive degeneration and deletion of dopaminergic neurons in the substantia nigra par compacta(SNpc).More and more studies have shown that neuroinflammation plays an important role in the pathogenesis of PD.Microglia is the important immune response cell in central nervous system.Studies have shown that dopaminergic neurons are susceptible to inflammation.The PET imaging results of PD patients have showed that the activation of microglia occurs in parallel to the loss of dopaminergic neurons,thus suggesting that the two processes are closely related.Therefore,the protection of DA neurons can be achieved by inhibiting the inflammatory response by anti-inflammatory drug,which will postpone the progress of PD and become a new strategy for PD treatment.Icariin(ICA),a flavonoid extract isolated from Epimedium,is the main active ingredient of Epimedium.Studies have shown that oral administration of ICA could produce icaritin(ICT)by the hydrolysis of intestinal flora in digestive tract.Many researchers have reported the neuroprotective effects of ICA.It can ameliorate the cognitive disorder of older rat and activate the quiescent neural stem cells of hippocampus.ICA could suppress apoptosis of hypothalamic neurons.Moreover,ICA has the apparently anti-inflammatory effect.Zeng KW reported that ICA could protect against lipopolysaccharide(LPS)-induced activation of primary cultured cortical microglia by inhibiting TAK1/IKK/NF-?B and JNK/p38 MAPK signaling pathway.However,the protective effect of ICA against inflammation-induced dopaminergic neuronal damage in nigrostriatal system and the precise underlying mechanisms remains unclear.ICT,as a hydrolytic derivative of ICA,has been confirmed to be a phytoestrogen and also play an estrogenic neuroprotective and anti-inflammatory effect.However,the upstream target molecule of ICA and ICT is still unclear.G protein-coupled estrogen receptor(GPER)is a member of the superfamily of 7-transmembrane G protein coupled receptor.Studies have shown that the rapid non-genomic effect of estrogen mediated by GPER is involved in the anti-inflammatory and neuroprotective effects of 17?-estradiol.After estrogen binding to GPER,the activated G protein interacts with adenylate cyclase,phospholipase C and growth factor receptor signaling pathways.Then estrogen rapidly regulates cell function and plays its biological roles.Studies also found that G protein-coupled estrogen receptor,widely expressed in the substantia nigra and striatum,is closely related to the protective effect of estrogen on dopaminergic neurons.Ma HR reported that ICA and ICT could stimulate the proliferation of breast cancer SKBr3 cells through GPER,epidermal growth factor receptor(EGFR)-induced mitogen-activated protein kinase(MAPK)signaling pathway.Our previous work revealed that ICT could protect against MPP+-induced damage in dopaminergic MES23.5 cells via the IGF-1R signaling pathway.In PD mice model,ICA could protect against MPTP-induced dopaminergic neuronal death and its possible mechanism might be related to the activation of MEK/ERK and PI3K/Akt signaling pathways.In view of the above research background,we wondered that whether GPER and IGF-1R-mediated signaling pathways were involved in the anti-inflammatory neuroprotective effects of ICA and ICT.In the first part of our study,BV2 cells were treated with LPS to establish an inflammatory cellular model.We explored the role of GPER and IGF-1R in anti-inflammatory mechanisms of ICA and ICT in vitro.In the second part,the inflammatory rat model of PD was established by the microinjection of LPS into substantia nigra.We aimed to further explore whether GPER and IGF-1R participated in the protection of ICA on doparminergic neurons by inhibiting microglial inflammation.1.The anti-inflammation effects of ICA and ICT in BV2 microglial cells via GPER and IGF-1RIn order to explore the anti-inflammatory effects of ICA and ICT via GPER and IGF-IR in BV2 microglial cells,the LPS-induced BV2 microglial cells were treated with ICA or ICT in the presence or absence of G15 or JB-1.The results are as follows:(1)LPS could remarkably enhance the m RNA expression of TNF-??IL-1??COX-2 and i NOS in BV2 cells(P<0.05).Different dosage of ICA(1,10,20,50 ?M)or ICT(1,10,20,50 ?M)pretreatment could inhibit the above inflammatory cytokines gene expression,and the most effective dosage is 10?M and 20?M(P<0.05,P<0.01,P<0.001).We chose the concentration of 10?M ICA and ICT in subsequent experiments.(2)The anti-inflammatory function of ICA and ICT on the m RNA expressions of COX-2?i NOS and IL-1? in BV2 microglial cells could be antagonized by GPER antagonist G15(P<0.05,P<0.01).(3)IGF-1R antagonist JB-1 could also abolished the anti-inflammatory effects of ICA and ICT against LPS-induced the up-regulation of the m RNA levels of COX-2?i NOS and IL-1? in BV2 microglial cells(P<0.05,P<0.01,P<0.001).(4)Treatment with ICA and ICT significantly inhibited the protein expressions of COX-2 and i NOS(P<0.01,P<0.001).These effects could be antagonized by G15 or JB-1(P<0.05,P<0.001).(5)LPS remarkably enhanced ERK,p38 and JNK phosphorylation level(P<0.05,P<0.01).Treatment with ICA and ICT could significantly attenuate these effects(P<0.05,P<0.01,P<0.001),while co-treatment with G15 or JB-1 antagonized the protective effects of ICA and ICT(P<0.05,P<0.01,P<0.001).(6)LPS remarkably enhanced I?B phosphorylation level(P<0.05,P<0.01).Treatment with ICA and ICT remarkably inhibited LPS-induced I?B phosphorylation level(P<0.01,P<0.001).These effects could be also blocked by G15 or JB-1(P<0.01).These results suggest that ICA and ICT could exert anti-inflammatory functions in BV2 microglia via GPER and IGF-1R-mediated signal pathways.2.The protective effect of ICA on dopaminergic neurons in rat substantia nigra by inhibiting microglial inflammatory response via GPER and IGF-1R.In the present study,rat inflammatory model of PD was established by unilateral microinjection of LPS into substantia nigra.Rats were administered with ICA(10 mg/kg)via gavage.The rotation behavior of rats was induced by intraperitoneal injection of apomorphine(APO).Using high performance liquid chromatography(HPLC),real time PCR and western blot,we systematically explored the molecular mechanism of ICA by which it protects dopaminergic neurons and inhibits inflammatory responses.The results are as follows:(1)Rotation behavior: APO-induced rat rotation was obviously increased in LPS inflammatory group(P<0.001);Compared with LPS treatment rats,ICA(10 mg/kg)treatment obviously reduced the APO-induced rotation behavior(P<0.001).This effect was antagonized by GPER antagonist G15 or IGF-1R antagonist JB-1(P<0.001).There was no significant rotation behavior in ICA treatment alone group.(2)The HPLC results showed that DA and its metabolites DOPAC,HVA content in striatum of the lesioned side were obviously reduced when compared with the control group(P<0.001);ICA treatment significantly ameliorated LPS-induced decrease of DA,HVA and DOPAC(P<0.05,P<0.001)in striatum of the lesioned side.These effects were antagonized by G15 or JB-1(P<0.05,P<0.01,P<0.001).There was no significant difference in DA,HVA and DOPAC content in ICA treatment alone group compared with control group.(3)The number of TH-immunoreactive neurons in the substantia nigra of the lesioned side was significantly lower than that of the unlesioned side with a survival rate of 37%.The expression of TH protein in the lesioned side of substantia nigra was also significantly lower than that of the control group(P<0.01,P<0.001).The numbers of TH immunoreactive neuron in SNpc as well as TH protein expression were significantly increased in ICA treatment group(P<0.05,P<0.01).The survival rate of TH immunoreactive neuron was more than 60%.This effect was antagonized by G15 or JB-1(P<0.05,P<0.01).There was no significant difference in TH-immunoreactive neuron number and TH protein expression in ICA treatment alone group compared with control group.(4)The results of immunohistochemisty showed that ICA treatment remarkably suppressed LPS-induced microglial activation in the lesioned side of SN.The inhibition of ICA on LPS-induced microglial over-activation was abolished by G15 or JB-1.There was no significant activation of microglia in ICA treatment alone group.(5)The real time RT-PCR results showed ICA remarkably suppressed microglial activation and decreased production of IL-1?,TNF-? and i NOS(P<0.01,P<0.001).Co-treatment with G15 or JB-1 abolished these effects(P<0.05,P<0.01).There was no significant difference in m RNA expression of IL-1?,TNF-? and i NOS in ICA treatment alone group compared with control group.(6)Compared with control group,the protein expressions of COX-2 and i NOS were significantly increased in LPS group(P<0.01).ICA significantly inhibited the LPS-induced protein expressions of COX-2 and i NOS(P<0.05,P<0.001).This effect could be antagonized by G15 or JB-1(P<0.05,P<0.01).There was no significant difference in COX-2 and i NOS expression in ICA treatment alone group compared with control group.(7)LPS intranigral injection remarkably enhanced ERK,p38 and JNK phosphorylation level(P<0.05,P<0.01).ICA treatment could significantly attenuate these effects(P<0.05,P<0.01).Moreover,co-treatment with G15 or JB-1 antagonized the protective effects of ICA(P<0.05).(8)LPS treatment down-regulated the IGF-1R protein expression in the lesioned side of SN(P<0.01).ICA treatment obviously increased the expression of IGF-1R compared to LPS group(P<0.01).This effect could be antagonized by G15 or JB-1(P<0.05).These results suggest that ICA may protect dopaminergic neurons by inhibiting microglial inflammation through the GPER and IGF-1R signaling pathways.Conclusions:Taken together,in in vitro study,ICA and ICT significantly inhibited LPS-induced inflammation in BV2 microglial cells.In in vivo study,ICA can protect the dopaminergic neurons in substantia nigra by inhibiting microglia inflammatory response.GPER and IGF-1R signaling pathways might be involved in the anti-inflammatory effects of ICA and ICT.This study provides a new experimental basis and strategy for the treatment of PD. |
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